Didier Clément

A genetic linkage map of Theobroma cacao L.

A linkage map of the cocoa genome comprising 193 loci has been constructed. These loci consist of 5 isozymes, 101 cDNA/RFLPs, 4 loci from genes of known function, 55 genomic DNA/RFLPs and 28 RAPDs. A population of 100 individuals derived from a cross between two heterozygous genotypes was used. Segregation analyses were performed with the JoinMap program. Ten linkage groups, which putatively correspond to the ten gametic chromosomes of cocoa, were identified. The map covers a total length of 759 cM with a 3.9 cM average distance between 2 markers. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios.

A meta-QTL analysis of disease resistance traits of Theobroma cacao L.

Theobroma cacao, is a tropical understorey tree that is a major economic resource to several tropical countries. However, the crop is under increased threat from several diseases that are responsible for 30% loss of harvest globally. Although QTL data related to the genetic determinism of disease resistance exist in cocoa, QTL mapping experiments are heterogeneous, thus making comparative QTL mapping essential for marker assisted selection (MAS). Sixteen QTL experiments were analysed, and the 76 QTLs detected were projected on a progressively established consensus map. Several hot spots, with QTLs related to different Phytophthora species and other diseases, were observed. The likely number of “real” QTLs was estimated by using a meta-analysis implemented in BioMercator software. There was a twofold reduction in average confidence interval observed when compared to the confidence interval of individual QTLs. This alternative approach confirms the existence of several sources of resistance to different diseases of cocoa which could be cumulated in new varieties to increase the sustainability of cocoa resistance using MAS strategies.

Discovery and mapping of a new expressed sequence tag-single nucleotide polymorphism and simple sequence repeat panel for large-scale genetic studies and breeding of Theobroma cacao L.

Theobroma cacao is an economically important tree of several tropical countries. Its genetic improvement is essential to provide protection against major diseases and improve chocolate quality. We discovered and mapped new expressed sequence tag-single nucleotide polymorphism (EST-SNP) and simple sequence repeat (SSR) markers and constructed a high-density genetic map. By screening 149 650 ESTs, 5246 SNPs were detected in silico, of which 1536 corresponded to genes with a putative function, while 851 had a clear polymorphic pattern across a collection of genetic resources. In addition, 409 new SSR markers were detected on the Criollo genome. Lastly, 681 new EST-SNPs and 163 new SSRs were added to the pre-existing 418 co-dominant markers to construct a large consensus genetic map. This high-density map and the set of new genetic markers identified in this study are a milestone in cocoa genomics and for marker-assisted breeding. The data are available at http://tropgenedb.cirad.fr.

Cacao breeding in Bahia, Brazil: strategies and results

Cacao was introduced in Bahia in 1756, becoming later the lar gest producer state in the country. In order to support the planting of cacao in the region, a breeding program was established by CEPEC at the beginning of the 1970s. For a long time, the program consisted in testing new hybrids (full-sibs) and r eleasing a mixture of the best ones to farmers. Lately, particularly after the witchesbroom arrival in the region, in 1989, recurrent breeding strategies were implemented, aiming mainly the development of clones. From 1993 to 2010, more than 500 progenies, accumulating 30 thousand trees, were developed by crossing many parents with resistance to witchesbroom, high yield and other traits. In this period, more than 500 clones were put in trials and 39 clones and 3 hybrids were released to farmers. In this paper the strategies and results achieved by the program are reviewed. Overall the program has good interface with pathology and genomic programs.

Development, characterization, validation, and mapping of SSRs derived from Theobroma cacao L.–Moniliophthora perniciosa interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F2 Sca 6 × ICS 1 population reference for WBD resistance.

A protocol for large scale genomic DNA isolation for cacao genetics analysis

Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the preservation of sample tissues for DNA extraction and for manual extraction of large number of samples using spheres. The integrity and concentration of the DNA by these methods were assessed and compared with conventional method using mortar. The best parameters in order to obtain a fine powder using spheres was the use of 4 lyophilized leaf disks (50 mg), a single steel ball of 6 mm in diameter, followed by 30 s of manual maceration. The quantity of DNA obtained was four times higher than the conventional method. The purity of the DNA obtained was satisfactory and proved to be amplifiable by PCR using SSR primers. The present approach is a reliable, rapid, simple and consistent DNA isolation method for cacao, compared to the conventional methods. The protocol greatly increases the efficiency of extraction and suggests an inexpensive and practical way of DNA isolation of cacao for large scale. Keywords : DNA extraction, cacao, spheres, lyophilized.

Identificação de resistência genética do cacaueiro à podridão-parda

The objective of this work was to compare methods for characterizing the resistance of cacao (Theobroma cacao) to black pod (Phytophthora palmivora). The methods of disease scores, intensity of infection index and disease index were evaluated. Leaf discs of 103 genotypes were infected with 0.2 mL of a suspension with 3x105 zoospores of P. palmivora per milliliter, and symptoms were evaluated by the three methods. Three evaluations (replicates) composed by 20 discs each were carried out for every genotype. The disease index method was the most efficient to characterize the cacao resistance to P. palmivora.

Genome size, cytogenetic data and transferability of EST-SSRs markers in wild and cultivated species of the genus #Theobroma# L. (Byttnerioideae, Malvaceae)

The genus Theobroma comprises several trees species native to the Amazon. Theobroma cacao L. plays a key economic role mainly in the chocolate industry. Both cultivated and wild forms are described within the genus. Variations in genome size and chromosome number have been used for prediction purposes including the frequency of interspecific hybridization or inference about evolutionary relationships. In this study, the nuclear DNA content, karyotype and genetic diversity using functional microsatellites (EST-SSR) of seven Theobroma species were characterized. The nuclear content of DNA for all analyzed Theobroma species was 1C = ~ 0.46 pg. These species presented 2n = 20 with small chromosomes and only one pair of terminal heterochromatic bands positively stained (CMA+/DAPI− bands). The small size of Theobroma ssp. genomes was equivalent to other Byttnerioideae species, suggesting that the basal lineage of Malvaceae have smaller genomes and that there was an expansion of 2C values in the more specialized family clades. A set of 20 EST-SSR primers were characterized for related species of Theobroma, in which 12 loci were polymorphic. The polymorphism information content (PIC) ranged from 0.23 to 0.65, indicating a high level of information per locus. Combined results of flow cytometry, cytogenetic data and EST-SSRs markers will contribute to better describe the species and infer about the evolutionary relationships among Theobroma species. In addition, the importance of a core collection for conservation purposes is highlighted.

Resistance gradient of black pod disease in cocoa and selection by leaf disk assay

To characterize the gradient of resistance to black pod disease experimentally, the leaf disk assay was applied to 217 cocoa genotypes. The parameters time interval, number of leaves evaluated per genotype and distribution of experimental treatments were evaluated. Significant differences between genotypes were found, resulting in the grouping in five homogenous groups (p < 0.01). In the assessments with the leaf disk test, five and seven days after inoculation, the F value was highest after seven days, clearly separating the resistant from susceptible genotypes. There was no difference between the clones behavior and the use of more than one box to group the genotypes (p = 0.56) and the use of different leaves of the same genotype (p = 0.08). The resistance gradient observed indicates variability enough for mapping and cocoa breeding in the population.

Deciphering the Theobroma cacao self-incompatibility system: from genomics to diagnostic markers for self-compatibility

Two different mechanisms controlling the Theobroma cacao self-incompatibility system were identified at two separate loci together with candidate genes differentially expressed between self-compatible/incompatible reactions, and diagnostic markers predicting self-compatible varieties were produced.